Zoonotic cutaneous leishmaniasis is an important health problem in Iran and a great economic burden on the health resources. In southern Iran (Fars Province), Meriones libycus was reported as the main reservoir of zoonotic cutaneous leishmaniasis in Arsanjan and Marvdasht cities, and Tatera indica and Gerbillus spp. were the reservoirs reported in Larestan and Kharameh districts. Because of an increase in human cutaneous leishmaniasis in Fars Province, this study was performed to identify the rodent hosts in this region. From April 2004 to April 2006, live traps were used to catch rodents in different parts of Fars Province. Fifty-seven Rattus norvegicus were caught and checked for Leishmania infection using a combination of microscopy, culture, nested polymerase chain reaction (PCR), and enzyme electrophoresis. One female R. norvegicus was found to be smear positive for amastigotes in Giemsa-stained skin sample of sole, and it was also culture positive for Leishmania. Results of PCR and isoenzyme electrophoresis indicated that this infected rodent was harboring Leishmania major. PCR was also positive for L. major in biopsy of soles, ear, liver, and spleen of 29 other R. norvegicus hosts that were negative in smears and cultures. There were no lesions seen in any parts of infected rodents' bodies. As L. major has not been previously reported in R. norvegicus in Iran or elsewhere, the rodent can be considered as a possible reservoir in transmission of the disease in Fars Province, and it should be brought into consideration when planning for preventive measures.